24th Feb 2006 Jane Lomax Gene Ontology tutorial Talk:Using the Gene Ontology (GO) for Expression Analysis Practical:Onto-Express analysis tool Talk: GO further Practical: AmiGO browser Practical: Annotation exercise

24th Feb 2006 Jane Lomax Using the Gene Ontology (GO) for Expression Analysis

24th Feb 2006 Jane Lomax GO for Expression Analysis What is GO? Structure of GO GO tools for microarray analysis Onto-Express tutorial

24th Feb 2006 Jane Lomax What is the Gene Ontology? Set of biological phrases (terms) which are applied to genes: –protein kinase –apoptosis –membrane

24th Feb 2006 Jane Lomax What is the Gene Ontology? Genes are linked, or associated, with GO terms by trained curators at genome databases –known as ‘gene associations’ or GO annotations Some GO annotations created automatically

24th Feb 2006 Jane Lomax gene -> GO term associated genes GO annotations GO database genome and protein databases

24th Feb 2006 Jane Lomax What is the Gene Ontology? Allows biologists to make inferences across large numbers of genes without researching each one individually

Copyright ©1998 by the National Academy of Sciences Eisen, Michael B. et al. (1998) Proc. Natl. Acad. Sci. USA 95,

24th Feb 2006 Jane Lomax GO structure GO isn’t just a flat list of biological terms terms are related within a hierarchy

24th Feb 2006 Jane Lomax GO structure gene A

24th Feb 2006 Jane Lomax GO structure This means genes can be grouped according to user-defined levels Allows broad overview of gene set or genome

24th Feb 2006 Jane Lomax How does GO work? GO is species independent –some terms, especially lower-level, detailed terms may be specific to a certain group e.g. photosynthesis –But when collapsed up to the higher levels, terms are not dependent on species

24th Feb 2006 Jane Lomax How does GO work? What does the gene product do? Where and when does it act? Why does it perform these activities? What information might we want to capture about a gene product?

24th Feb 2006 Jane Lomax GO structure GO terms divided into three parts: –cellular component –molecular function –biological process

24th Feb 2006 Jane Lomax Cellular Component where a gene product acts

24th Feb 2006 Jane Lomax Cellular Component

24th Feb 2006 Jane Lomax Cellular Component

24th Feb 2006 Jane Lomax Cellular Component Enzyme complexes in the component ontology refer to places, not activities.

24th Feb 2006 Jane Lomax Molecular Function activities or “ jobs ” of a gene product glucose-6-phosphate isomerase activity

24th Feb 2006 Jane Lomax Molecular Function insulin binding insulin receptor activity

24th Feb 2006 Jane Lomax Molecular Function drug transporter activity

24th Feb 2006 Jane Lomax Molecular Function A gene product may have several functions; a function term refers to a single reaction or activity, not a gene product. Sets of functions make up a biological process.

24th Feb 2006 Jane Lomax Biological Process a commonly recognized series of events cell division

24th Feb 2006 Jane Lomax Biological Process transcription

24th Feb 2006 Jane Lomax Biological Process regulation of gluconeogenesis

24th Feb 2006 Jane Lomax Biological Process limb development

24th Feb 2006 Jane Lomax Biological Process courtship behavior

24th Feb 2006 Jane Lomax Ontology Structure Terms are linked by two relationships –is-a  –part-of 

24th Feb 2006 Jane Lomax Ontology Structure cell membrane chloroplast mitochondrial chloroplast membrane is-a part-of

24th Feb 2006 Jane Lomax Ontology Structure Ontologies are structured as a hierarchical directed acyclic graph (DAG) Terms can have more than one parent and zero, one or more children

24th Feb 2006 Jane Lomax Ontology Structure cell membrane chloroplast mitochondrial chloroplast membrane Directed Acyclic Graph (DAG) - multiple parentage allowed

24th Feb 2006 Jane Lomax Anatomy of a GO term id: GO: name: gluconeogenesis namespace: process def: The formation of glucose from noncarbohydrate precursors, such as pyruvate, amino acids and glycerol. [ exact_synonym: glucose biosynthesis xref_analog: MetaCyc:GLUCONEO-PWY is_a: GO: is_a: GO: unique GO ID term name definition synonym database ref parentage ontology

24th Feb 2006 Jane Lomax GO tools GO resources are freely available to anyone to use without restriction –Includes the ontologies, gene associations and tools developed by GO Other groups have used GO to create tools for many purposes:

24th Feb 2006 Jane Lomax GO tools Affymetrix also provide a Gene Ontology Mining Tool as part of their NetAffx™ Analysis Center which returns GO terms for probe sets

24th Feb 2006 Jane Lomax GO tools Many tools exist that use GO to find common biological functions from a list of genes:

24th Feb 2006 Jane Lomax GO tools Most of these tools work in a similar way: –input a gene list and a subset of ‘interesting’ genes –tool shows which GO categories have most interesting genes associated with them i.e. which categories are ‘enriched’ for interesting genes –tool provides a statistical measure to determine whether enrichment is significant

24th Feb 2006 Jane Lomax Microarray process Treat samples Collect mRNA Label Hybridize Scan Normalize Select differentially regulated genes Understand the biological phenomena involved

24th Feb 2006 Jane Lomax Traditional analysis Gene 1 Apoptosis Cell-cell signaling Protein phosphorylation Mitosis … Gene 2 Growth control Mitosis Oncogenesis Protein phosphorylation … Gene 3 Growth control Mitosis Oncogenesis Protein phosphorylation … Gene 4 Nervous system Pregnancy Oncogenesis Mitosis … Gene 100 Positive ctrl. of cell prolif Mitosis Oncogenesis Glucose transport …

24th Feb 2006 Jane Lomax Traditional analysis gene by gene basis requires literature searching time-consuming

24th Feb 2006 Jane Lomax Using GO annotations But by using GO annotations, this work has already been done for you! GO: : apoptosis

24th Feb 2006 Jane Lomax Grouping by process Apoptosis Gene 1 Gene 53 Mitosis Gene 2 Gene 5 Gene45 Gene 7 Gene 35 … Positive ctrl. of cell prolif. Gene 7 Gene 3 Gene 12 … Growth Gene 5 Gene 2 Gene 6 … Glucose transport Gene 7 Gene 3 Gene 6 …

24th Feb 2006 Jane Lomax GO for microarray analysis Annotations give ‘function’ label to genes Ask meaningful questions of microarray data e.g. –genes involved in the same process, same/different expression patterns?

24th Feb 2006 Jane Lomax Using GO in practice statistical measure –how likely your differentially regulated genes fall into that category by chance microarray 1000 genes experiment100 genes differentially regualted mitosis – 80/100 apoptosis – 40/100 p. ctrl. cell prol. – 30/100 glucose transp. – 20/100

24th Feb 2006 Jane Lomax Using GO in practice However, when you look at the distribution of all genes on the microarray: ProcessGenes on array # genes expected in occurred 100 random genes mitosis 800/ apoptosis 400/ p. ctrl. cell prol. 100/ glucose transp. 50/

24th Feb 2006 Jane Lomax The tutorial Analysing microarray data using GO with Onto-Express

24th Feb 2006 Jane Lomax The tutorial - Onto-Express

24th Feb 2006 Jane Lomax Onto-Express walkthrough